Gifu anaerobic medium

Clostridium difficile (CD) toxigenic strains ATCC9689 and 630 [35 (link)] were used; both strains produce toxins A and B. The Bacteroides species used were B. thetaiotaomicron (strain VPI-5482 and seven clinical isolates), B. fragilis (NCTC9343 and YCH46 [49 (link)]), B. ovatus ATCC8483, B. uniformis ATCC8492, and B. vulgatus ATCC8482. Bifidobacterium longum JCM1217, Clostridium ramosum JCM1298, and Escherichia coli strain DH5α were also tested as other representative intestinal bacteria. All bacterial strains used were grown anaerobically in GAM (Gifu anaerobic medium, Nissui Pharmaceutical Co., Tokyo, Japan) broth or on GAM agar plates at 37 °C. Anaerobic cultivation was performed using an anaerobic chamber (Forma Scientific model 1025) conditioned with mixed gas (CO₂, 80%; N₂, 10%; H₂, 10%).

The cryostocks of these bacterial strains were inoculated in one of these media: BHI, Gifu anaerobic medium (Nissui Pharmaceutical, Tokyo, Japan), Man, Rogosa & Sharpe broth (Oxoid Ltd, Basingstoke, United Kingdom), or Standard feed (ProDigest BVBA, Ghent, Belgium) that has been extensively used in the in vitro gut fermentor-simulator of the Human Intestinal Microbial Ecosystem (SHIME; Prodigest BVBA, Ghent, Belgium) (20 (link), 26 (link), 27 (link)). Total cell counting was performed on the second passage using an impedance flow cytometer BactoBox (SBT Instruments, Herlev, Denmark). For each of the bacterial cultures, serial dilution series were conducted by using 6 mL vials containing a volume of 3 mL 1/20 phosphate-buffered saline buffer until the final concentration was within the detection range (10⁴–10⁶ bacteria/mL). Simultaneously, all the bacterial cultures were diluted 10 times and subjected to OD₆₀₀ measurement using a microplate Epoch 2 spectrophotometer (BioTek, Winooski, Vermont, USA).

A probiotic lactic acid bacterium, Lactobacillus gasseri JCM1131^T (=DSM20243^T=ATCC33323^T), was obtained from the Japan Collection of Microorganisms (RIKEN BRC, Tsukuba, Japan). This strain was cultivated using Gifu anaerobic medium (GAM, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and de Man–Rogosa–Sharpe medium (MRS, Difco Laboratories, Detroit, MI, USA) with headspace gas of N₂/CO₂ (80:20, v/v) at 37 °C under anaerobic conditions. Escherichia coli strain BL21 (DE3) Champion^TM21 (SMOBIO Technologies, Hsinchu City, Taiwan) was used for heterologous expression experiments. E. coli was cultured in LB broth supplemented with 50 μg/mL kanamycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 37 °C with shaking.

Methicillin-susceptible Staphylococcus aureus (MSSA) ATCC25923 and methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33,591 were aerobically incubated at 37 °C in Luria–Bertani medium (LB, Nippon Becton Dickinson Company, Tokyo, Japan). Porphyromonas gingivalis W83 was anaerobically incubated at 37 °C in Gifu anaerobic medium (GAM, Nissui, Tokyo, Japan). Each culture (20 µL) prepared to an optical density of 1.5 at 600 nm were appropriately incubated with various concentrations of synthesized compounds in 200 µL of culture medium at 37 °C for 24 h in 96-well plate (Thermo scientific, MA, USA). Compounds were dissolved in DMSO (Wako, Osaka, Japan). The degree of turbidity in the broth culture was measured at absorbance 600 nm using microplate reader (Thermo scientific, MA, USA).

V. parvula JCM 12972 and F. nucleatum subspecies polymorphum JCM 12990 were cultured anaerobically under 80% nitrogen, 10% hydrogen, and 10% carbon dioxide at 37 °C, using modified GAM (Gifu anaerobic medium; Nissui Pharmaceutical, Tokyo, Japan).

A Bio Jr.8 fermenter (ABLE, Tokyo, Japan) comprising eight parallel and independent anaerobic culturing vessels was used for fecal sample cultivation as described previously^{49 (link)}. Briefly, 0.5 g of fecal samples were suspended in 2 mL of PBS buffer (nacalai tesque, Kyoto, Japan). Each vessel containing 100 mL of Gifu anaerobic medium (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) was inoculated with either 100 μL of fecal suspension alone or with 0.3% (3 g/L) of CSs and then cultivated anaerobically at 37 °C (n = 10). The culture broth was stirred at 300 rpm and continuously purged with an anaerobic gas mixture (N₂:CO₂ = 80:20) to maintain anaerobic conditions. After 48 h of cultivation, the culture broths were collected and used for subsequent analyses.